The ability to introduce drugs, genes, nucleic acids, and/or imaging agents into living cells is critical to drug design and delivery, as well as many cell biology and genetic modification protocols;however, currently available physical and reagent-based techniques are inadequate for applications requiring transfection of difficult-to-transfect cells (e.g., primary and stem cells). For this reason, transfection of nucleic acids into cells has become a significant challenge in the development of RNAi therapies and stem cell clinical applications. The technology that is the subject of this project proposal has demonstrated the potential to significantly impact these areas by enabling investigations of difficult-to-transfect cells, which are not currently feasible. The proposed work addresses this challenge through development of a microfabricated technology that enables treatment of arbitrarily sized cell populations on a cell-by-cell basis. STEAM (Single-sample Treatment via Electrosonic Actuation Microarray) ejects biological cells through microscopic nozzles with incorporated electroporation electrodes, thereby opening pores by concurrent mechanical and electrical disruption of the cell membrane. The parallel microarray format is scalable to accommodate discrete sample volumes from ~100 nl to tens of ml;however, in continuous-flow mode, the same device can rapidly process cells at 1 to 100 million cells per second. The critical advantage of STEAM is the uniformity of treatment experienced by each cell in a population, which is the key to achieving high transfection efficiency. During the SBIR Phase I project a prototype STEAM device demonstrated successful treatment of laboratory established cell lines. Device operating parameters were optimized using a small fluorescent molecule to evaluate uptake and cell viability. In addition, STEAM achieved trasfection efficiencies of 80% (mechanical poration) and >90% (mechanical + electroporation) for GFP-encoding plasmid into HEK293 cells with cell viability >70%, which is on par with lipofection and the best commercially available electroporation systems. The primary objectives of this SBIR Phase II project are further device refinement and optimization towards development of a production prototype and direct comparison with available physical and reagent-based techniques for transfection of difficult cells. To achieve these objectives, (1) a stand-alone STEAM system with disposable cartridge-based sample handling and on-board electronic control of both mechanical and electroporation parameters will be developed, and (2) a direct comparison of STEAM, commercial electroporation systems, lipofectamine-mediated transfection, and lentiviral gene transfer in difficult cells (including primary cancer stem cells from glioblastoma multiforme) will be performed. PUBLIC HEALTH RELEVANCE: Development of the STEAM (Single-sample Treatment via Electrosonic Actuation Microarray) platform will address the current need for alternative gene transfer solutions for use with difficult-to-transfect cells (e.g., primary and stem cells). The lack of successful commercial gene transfer solutions limits research in the life sciences and biomedical fields. STEAM addresses the need for effective, high-throughput, and scalable techniques to achieve transfection of difficult cells.